title: Structure-based optimization of FDA-approved drug methylene blue as a c-myc G-quadruplex DNA stabilizer.
abstract: G-quadruplexes are non-canonical DNA secondary structures putatively present in the promoter regions of oncogenes in the human genome. The targeting of promoter G-quadruplex structures to repress oncogene transcription represents a potential anticancer strategy. Here, we have used high-throughput virtual screening to identify FDA-approved drug methylene blue (MB) as a promising scaffold for binding the c-myc oncogene G-quadruplex DNA. Based on molecular docking analysis of MB to the c-myc G-quadruplex, we designed and screened 50 MB derivatives containing side chains that could interact with the G-quadruplex grooves. As a proof-of-concept, the highest-scoring compounds were synthesized and the interactions with the c-myc G-quadruplex were investigated using the FID assay. The results showed that the methylene blue derivatives 6a-c were able to bind to the c-myc G-quadruplex with greater binding affinity compared to the known G-quadruplex binding ligand, crystal violet. The activity of the most potent compound identified from the FID assay, 6b, as an inhibitor for polymerase-drive DNA extension was examined using a PCR-stop assay and compared against that of the parent compound methylene blue. The results of the PCR-stop assay showed that the addition of the side chain improved the activity of the derivatives as an inhibitor compared to the parent compound. The MB derivative 6b was shown to be highly selective towards c-myc G-quadruplex over double-stranded DNA and other biologically relevant G-quadruplexes using UV-visible spectroscopy and mass spectrometry, respectively. The MB derivative 6b could induce or stabilize c-myc G-quadruplex formation in both cell-free and cellular biological models, and displayed higher cytoxicity against human hepatocarcinoma cells compared to the parent compound, MB.
authors.affiliation: Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.
authors.forename: Daniel Shiu-Hin
authors.initials: DS
authors.lastname: Chan
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.forename: Maria Hiu-Tung
authors.initials: MH
authors.lastname: Kwan
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cheng
authors.forename: Paul
authors.initials: P
authors.lastname: Lee
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Bai
authors.forename: Zhi-Hong
authors.initials: ZH
authors.lastname: Jiang
authors.forename: Chun-Yuen
authors.initials: CY
authors.lastname: Wong
authors.forename: Wang-Fun
authors.initials: WF
authors.lastname: Fong
authors.forename: Chung-Hang
authors.initials: CH
authors.lastname: Leung
authors.forename: Dik-Lung
authors.initials: DL
authors.lastname: Ma
mesh_terms: D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D017277:Drug Approval; D054856:G-Quadruplexes / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D016259:Genes, myc; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008751:Methylene Blue / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D056004:Molecular Dynamics Simulation; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship
chemical_list: C560212:3,7-bis((4-bromophenylethyl)(methyl)amino)phenothiazin-5-ium; D000970:Antineoplastic Agents; D008156:Luciferases; D008751:Methylene Blue